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1.  Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT) 
Transgenic Research  2012;22(4):709-723.
Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using minicircles in RMCE in fibroblasts with all four acceptor loci and followed by SCNT, we produced piglets with a single copy of a transgene incorporated into one of the transcriptionally active acceptor loci. The transgene, consisting of a cDNA of the Alzheimer’s disease-causing gene PSEN1M146I driven by an enhanced human UbiC promoter, had an expression profile in various tissues similar to that of the GFP marker gene. The results show that RMCE can be done in a pre-selected transcriptionally active acceptor locus for targeted transgenesis in pigs.
Electronic supplementary material
The online version of this article (doi:10.1007/s11248-012-9671-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s11248-012-9671-6
PMCID: PMC3712138  PMID: 23111619
RMCE; Sleeping Beauty; SCNT; Targeted transgenesis; Transgenic pigs; Disease models
2.  Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 and α2 Integrin 
PLoS ONE  2012;7(5):e36658.
Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage-induced inflammation and should be of great potential in studies aiming at the development and refinement of topical therapies for cutaneous inflammation including psoriasis.
doi:10.1371/journal.pone.0036658
PMCID: PMC3349713  PMID: 22590584
3.  Metabolomic phenotyping of a cloned pig model 
BMC Physiology  2011;11:14.
Background
Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs.
Results
The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established.
Conclusions
From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.
doi:10.1186/1472-6793-11-14
PMCID: PMC3174869  PMID: 21859467

Results 1-3 (3)